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`øÎÛÐÎ×ÒÌÛÜ÷ ßÎÝØ ÑÐØÌØßÔÓÑÔ ñ ÊÑÔ ïïèô ÒÑÊ îððð
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`îððð ß³»®·½¿² Ó»¼·½¿´ ß±½·¿¬·±²ò ß´´ ®·¹¸¬ ®»»®ª»¼ò
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`6-month biopsy specimens were obtained and similarly fro-
`êó³±²¬¸ ¾·±°§ °»½·³»² ©»®» ±¾¬¿·²»¼ ¿²¼ ·³·´¿®´§ º®±ó
`zen. Six-micrometer sections were taken from each block,
`¦»²ò Í·¨ó³·½®±³»¬»® »½¬·±² ©»®» ¬¿µ»² º®±³ »¿½¸ ¾´±½µô
`mounted on gelatin-coated slides, and processed for im-
`³±«²¬»¼ ±² ¹»´¿¬·²ó½±¿¬»¼ ´·¼»ô ¿²¼ °®±½»»¼ º±® ·³ó
`munohistochemical analysis. Sectioning of tissue blocks and
`³«²±¸·¬±½¸»³·½¿´ ¿²¿´§·ò Í»½¬·±²·²¹ ±º ¬·«» ¾´±½µ ¿²¼
`immunohistochemical experiments were performed as pairs
`·³³«²±¸·¬±½¸»³·½¿´ »¨°»®·³»²¬ ©»®» °»®º±®³»¼ ¿ °¿·®
`of biopsies, pretreatment and posttreatment, to minimize
`±º ¾·±°·»ô °®»¬®»¿¬³»²¬ ¿²¼ °±¬¬®»¿¬³»²¬ô ¬± ³·²·³·¦»
`differences due to experimental conditions.
`¼·ºº»®»²½» ¼«» ¬± »¨°»®·³»²¬¿´ ½±²¼·¬·±²ò
`
`IMMUNOHISTOCHEMICAL ANALYSIS
`×ÓÓËÒÑØ×ÍÌÑÝØÛÓ×ÝßÔ ßÒßÔÇÍ×Í
`
`Immunohistochemical staining for lymphocytic markers as
`׳³«²±¸·¬±½¸»³·½¿´ ¬¿·²·²¹ º±® ´§³°¸±½§¬·½ ³¿®µ»® ¿
`well as lymphocyte activation markers was conducted us-
`©»´´ ¿ ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µ»® ©¿ ½±²¼«½¬»¼ «ó
`ing monoclonal antibodies to CD3 (PharMingen, San Diego,
`·²¹ ³±²±½´±²¿´ ¿²¬·¾±¼·» ¬± ÝÜí øи¿®Ó·²¹»²ô Í¿² Ü·»¹±ô
`Calif), CD4 (Becton—Dickinson, Sanjose, Calif), CD8 (Bec-
`Ý¿´·º÷ô ÝÜì øÞ»½¬±²óÜ·½µ·²±²ô Í¿² Ö±»ô Ý¿´·º÷ô ÝÜè øÞ»½ó
`ton—Dickinson, Sanjose), CD1 1a (PharMingen, San Diego),
`¬±²óÜ·½µ·²±²ô Í¿² Ö±»÷ô ÝÜïï¿ øи¿®Ó·²¹»²ô Í¿² Ü·»¹±÷ô
`and HLA—DR (PharMingen). Cryostat sections were fixed
`¿²¼ ØÔßóÜÎ øи¿®Ó·²¹»²÷ò Ý®§±¬¿¬ »½¬·±² ©»®» º·¨»¼
`in cold acetone (—20°C) for 3 minutes and air dried at room
`·² ½±´¼ ¿½»¬±²» ø"îðWÝ÷ º±® í ³·²«¬» ¿²¼ ¿·® ¼®·»¼ ¿¬ ®±±³
`temperature for 30 to 45 minutes. They were then rinsed
`¬»³°»®¿¬«®» º±® í𠬱 ìë ³·²«¬»ò ̸»§ ©»®» ¬¸»² ®·²»¼
`in 3 changes of phosphate-buffered saline (PBS) and incu-
`·² í ½¸¿²¹» ±º °¸±°¸¿¬»ó¾«ºº»®»¼ ¿´·²» øÐÞÍ÷ ¿²¼ ·²½«ó
`bated in PBS with 1% bovine serum albumin (BSA) (Sigma
`¾¿¬»¼ ·² ÐÞÍ ©·¬¸ ïû ¾±ª·²» »®«³ ¿´¾«³·² øÞÍß÷ øÍ·¹³¿
`Chemical Co, St Louis, Mo) for 10 minutes. Sections were
`ݸ»³·½¿´ ݱô ͬ Ô±«·ô Ó±÷ º±® ïð ³·²«¬»ò Í»½¬·±² ©»®»
`incubated for 1 hour at room temperature in primary an-
`·²½«¾¿¬»¼ º±® ï ¸±«® ¿¬ ®±±³ ¬»³°»®¿¬«®» ·² °®·³¿®§ ¿²ó
`tibodies at concentrations derived empirically: CD3, 1.0
`¬·¾±¼·» ¿¬ ½±²½»²¬®¿¬·±² ¼»®·ª»¼ »³°·®·½¿´´§æ ÝÜíô ïòð
`ug/mL; CD4, 5.0 ug/mL; CD8, 2.5 ug/mL; CD11a, 10.0
`¡¹ñ³Ôå ÝÜìô ëòð ¡¹ñ³Ôå ÝÜèô îòë ¡¹ñ³Ôå ÝÜïï¿ô ïðòð
`ug/mL; and HLA—DR, 1.0 ug/mL. Sections were rinsed in
`¡¹ñ³Ôå ¿²¼ ØÔßóÜÎô ïòð ¡¹ñ³Ôò Í»½¬·±² ©»®» ®·²»¼ ·²
`PBS alone, followed by 10 minutes in PBS with 1% BSA be-
`ÐÞÍ ¿´±²»ô º±´´±©»¼ ¾§ ïð ³·²«¬» ·² ÐÞÍ ©·¬¸ ïû ÞÍß ¾»ó
`fore incubation for 1 hour at room temperature in the sec-
`º±®» ·²½«¾¿¬·±² º±® ï ¸±«® ¿¬ ®±±³ ¬»³°»®¿¬«®» ·² ¬¸» »½ó
`ondary antibody, fluorescein isothiocyanate—conjugated Af-
`±²¼¿®§ ¿²¬·¾±¼§ô º´«±®»½»·² ·±¬¸·±½§¿²¿¬»o½±²¶«¹¿¬»¼ ߺó
`finipure Donkey Anti-Mouse IgG (]ackson Immunoresearch,
`º·²·°«®» ܱ²µ»§ ß²¬·óÓ±«» ×¹Ù øÖ¿½µ±² ׳³«²±®»»¿®½¸ô
`West Grove, Pa) at a dilution of 1/50. Sections were then
`É»¬ Ù®±ª»ô п÷ ¿¬ ¿ ¼·´«¬·±² ±º ïñëðò Í»½¬·±² ©»®» ¬¸»²
`rinsed in PBS, mounted in Vectashield (Vector Labs, Bur-
`®·²»¼ ·² ÐÞÍô ³±«²¬»¼ ·² Ê»½¬¿¸·»´¼ øÊ»½¬±® Ô¿¾ô Þ«®ó
`lingame, Calif), cover-slipped, and viewed under a micro-
`´·²¹¿³»ô Ý¿´·º÷ô ½±ª»®ó´·°°»¼ô ¿²¼ ª·»©»¼ «²¼»® ¿ ³·½®±ó
`scope (Eclipse E800; Nikon, Melville, NY) interfaced with
`½±°» øÛ½´·°» Ûèððå Ò·µ±²ô Ó»´ª·´´»ô ÒÇ÷ ·²¬»®º¿½»¼ ©·¬¸
`a digital camera (Spot Digital Camera; Diagnostic Instru-
`¿ ¼·¹·¬¿´ ½¿³»®¿ øÍ°±¬ Ü·¹·¬¿´ Ý¿³»®¿å Ü·¿¹²±¬·½ ײ¬®«ó
`ments Inc, Micro Video Instruments, Avon, Mass). Sec-
`³»²¬ ײ½ô Ó·½®± Ê·¼»± ײ¬®«³»²¬ô ߪ±²ô Ó¿÷ò Í»½ó
`ondary antibody controls omitting the primary antibody
`±²¼¿®§ ¿²¬·¾±¼§ ½±²¬®±´ ±³·¬¬·²¹ ¬¸» °®·³¿®§ ¿²¬·¾±¼§
`for all biopsy specimens for each immunohistochemical
`º±® ¿´´ ¾·±°§ °»½·³»² º±® »¿½¸ ·³³«²±¸·¬±½¸»³·½¿´
`analysis were run.
`¿²¿´§· ©»®» ®«²ò
`Three separate images were acquired for each anti-
`̸®»» »°¿®¿¬» ·³¿¹» ©»®» ¿½¯«·®»¼ º±® »¿½¸ ¿²¬·ó
`body and biopsy specimen under a X20 objective using a
`¾±¼§ ¿²¼ ¾·±°§ °»½·³»² «²¼»® ¿ íîð ±¾¶»½¬·ª» «·²¹ ¿
`Spot acquisition program (Diagnostic Instruments Inc). The
`Í°±¬ ¿½¯«··¬·±² °®±¹®¿³ øÜ·¿¹²±¬·½ ײ¬®«³»²¬ ײ½÷ò ̸»
`first field selected for imaging was the field with the high-
`º·®¬ º·»´¼ »´»½¬»¼ º±® ·³¿¹·²¹ ©¿ ¬¸» º·»´¼ ©·¬¸ ¬¸» ¸·¹¸ó
`est number of positive cells, followed by images to the left
`»¬ ²«³¾»® ±º °±·¬·ª» ½»´´ô º±´´±©»¼ ¾§ ·³¿¹» ¬± ¬¸» ´»º¬
`
`and right of that area. In this manner the entire biopsy area
`¿²¼ ®·¹¸¬ ±º ¬¸¿¬ ¿®»¿ò ײ ¬¸· ³¿²²»® ¬¸» »²¬·®» ¾·±°§ ¿®»¿
`was usually captured.
`©¿ ««¿´´§ ½¿°¬«®»¼ò
`
`COUNTING PROCEDURE
`ÝÑËÒÌ×ÒÙ ÐÎÑÝÛÜËÎÛ
`
`Measurement of the entire area of epithelium and stroma
`Ó»¿«®»³»²¬ ±º ¬¸» »²¬·®» ¿®»¿ ±º »°·¬¸»´·«³ ¿²¼ ¬®±³¿
`(substantia propria) was achieved by tracing the area us-
`ø«¾¬¿²¬·¿ °®±°®·¿÷ ©¿ ¿½¸·»ª»¼ ¾§ ¬®¿½·²¹ ¬¸» ¿®»¿ «ó
`ing the lasso tool under the Adobe Photoshop computer
`·²¹ ¬¸» ´¿± ¬±±´ «²¼»® ¬¸» ß¼±¾» 豬±¸±° ½±³°«¬»®
`program (Adobe Systems Inc, Sanjose, Calif). The total data
`°®±¹®¿³ øß¼±¾» ͧ¬»³ ײ½ô Í¿² Ö±»ô Ý¿´·º÷ò ̸» ¬±¬¿´ ¼¿¬¿
`area, measured in pixels, was acquired through the “Im-
`¿®»¿ô ³»¿«®»¼ ·² °·¨»´ô ©¿ ¿½¯«·®»¼ ¬¸®±«¹¸ ¬¸» v׳ó
`age: Histogram” command in Photoshop. Two indepen-
`¿¹»æ Ø·¬±¹®¿³f ½±³³¿²¼ ·² 豬±¸±°ò Ì©± ·²¼»°»²ó
`dent counts were recorded for cells positive for each anti-
`¼»²¬ ½±«²¬ ©»®» ®»½±®¼»¼ º±® ½»´´ °±·¬·ª» º±® »¿½¸ ¿²¬·ó
`body within the traced area. Cells per unit area of pixels
`¾±¼§ ©·¬¸·² ¬¸» ¬®¿½»¼ ¿®»¿ò Ý»´´ °»® «²·¬ ¿®»¿ ±º °·¨»´
`were adjusted to real unit area or cells per millimeter squared
`©»®» ¿¼¶«¬»¼ ¬± ®»¿´ «²·¬ ¿®»¿ ±® ½»´´ °»® ³·´´·³»¬»® ¯«¿®»¼
`of real tissue area, based on 28.346 pixels per centimeter
`±º ®»¿´ ¬·«» ¿®»¿ô ¾¿»¼ ±² îèòíìê °·¨»´ °»® ½»²¬·³»¬»®
`in Photoshop and the fact that 1 mm equals 67.8 cm equals
`·² 豬±¸±° ¿²¼ ¬¸» º¿½¬ ¬¸¿¬ ï ³³ »¯«¿´ êéòè ½³ »¯«¿´
`1922 pixels at X20 magnification on the Nikon micro-
`ïçîî °·¨»´ ¿¬ íî𠳿¹²·º·½¿¬·±² ±² ¬¸» Ò·µ±² ³·½®±ó
`scope. Data were recorded as cells per millimeter squared
`½±°»ò Ü¿¬¿ ©»®» ®»½±®¼»¼ ¿ ½»´´ °»® ³·´´·³»¬»® ¯«¿®»¼
`for all markers, and statistical analysis was based on these
`º±® ¿´´ ³¿®µ»®ô ¿²¼ ¬¿¬·¬·½¿´ ¿²¿´§· ©¿ ¾¿»¼ ±² ¬¸»»
`measurements.
`³»¿«®»³»²¬ò
`
`STATISTICAL METHODS
`ÍÌßÌ×ÍÌ×ÝßÔ ÓÛÌØÑÜÍ
`
`Baseline characteristics were tabulated and summarized by
`Þ¿»´·²» ½¸¿®¿½¬»®·¬·½ ©»®» ¬¿¾«´¿¬»¼ ¿²¼ «³³¿®·¦»¼ ¾§
`treatment groups. Overall differences among treatment
`¬®»¿¬³»²¬ ¹®±«°ò Ѫ»®¿´´ ¼·ºº»®»²½» ¿³±²¹ ¬®»¿¬³»²¬
`groups were tested using a 2-way analysis of variance
`¹®±«° ©»®» ¬»¬»¼ «·²¹ ¿ î󩿧 ¿²¿´§· ±º ª¿®·¿²½»
`(ANOVA) for continuous variables and the Fisher exact test
`øßÒÑÊß÷ º±® ½±²¬·²«±« ª¿®·¿¾´» ¿²¼ ¬¸» Ú·¸»® »¨¿½¬ ¬»¬
`for categorical variables.
`º±® ½¿¬»¹±®·½¿´ ª¿®·¿¾´»ò
`Percent changes in the number of cells expressing
`л®½»²¬ ½¸¿²¹» ·² ¬¸» ²«³¾»® ±º ½»´´ »¨°®»·²¹
`lymphocytic and/or lymphocyte activation markers were
`´§³°¸±½§¬·½ ¿²¼ñ±® ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µ»® ©»®»
`summarized using descriptive statistics (ie, sample size,
`«³³¿®·¦»¼ «·²¹ ¼»½®·°¬·ª» ¬¿¬·¬·½ ø·»ô ¿³°´» ·¦»ô
`mean, SD, minimum, maximum, and median). A 1-way
`³»¿²ô ÍÜô ³·²·³«³ô ³¿¨·³«³ô ¿²¼ ³»¼·¿²÷ò ß ï󩿧
`ANOVA with main effect for treatment was used to test
`ßÒÑÊß ©·¬¸ ³¿·² »ºº»½¬ º±® ¬®»¿¬³»²¬ ©¿ «»¼ ¬± ¬»¬
`for differences in percent change from baseline and
`º±® ¼·ºº»®»²½» ·² °»®½»²¬ ½¸¿²¹» º®±³ ¾¿»´·²» ¿²¼
`ratios among treatment groups by visit. If the test for
`®¿¬·± ¿³±²¹ ¬®»¿¬³»²¬ ¹®±«° ¾§ ª··¬ò ׺ ¬¸» ¬»¬ º±®
`among-group differences in main effect was significant,
`¿³±²¹ó¹®±«° ¼·ºº»®»²½» ·² ³¿·² »ºº»½¬ ©¿ ·¹²·º·½¿²¬ô
`then all 3 pairwise comparisons were made. Within-
`¬¸»² ¿´´ í °¿·®©·» ½±³°¿®·±² ©»®» ³¿¼»ò É·¬¸·²ó
`group changes from baseline were analyzed by the
`¹®±«° ½¸¿²¹» º®±³ ¾¿»´·²» ©»®» ¿²¿´§¦»¼ ¾§ ¬¸»
`paired t test method.
`°¿·®»¼ ¬ ¬»¬ ³»¬¸±¼ò
`The same analysis was performed on Sjogren and
`̸» ¿³» ¿²¿´§· ©¿ °»®º±®³»¼ ±² Ͷ±X ¹®»² ¿²¼
`non-Sjogren subpopulations, excluding the 0.1% CsA
`²±²óͶ±X ¹®»² «¾°±°«´¿¬·±²ô »¨½´«¼·²¹ ¬¸» ðòïû Ýß
`treatment group in which there was only 1 patient in the
`¬®»¿¬³»²¬ ¹®±«° ·² ©¸·½¸ ¬¸»®» ©¿ ±²´§ ï °¿¬·»²¬ ·² ¬¸»
`Sjogren subset.
`Ͷ±X ¹®»² «¾»¬ò
`
`(CD1 1a and HLA—DR) to further understand the under-
`øÝÜïï¿ ¿²¼ ØÔßóÜÎ÷ ¬± º«®¬¸»® «²¼»®¬¿²¼ ¬¸» «²¼»®ó
`´§·²¹ ³»½¸¿²·³ ±º Ýß ¬®»¿¬³»²¬ò
`lying mechanism of CsA treatment.
`
`ÎÛÍËÔÌÍ
`
`¬®»¿¬³»²¬ ©·¬¸ »·¬¸»® ½±²½»²¬®¿¬·±² ±º Ýßò ̸» ±²´§ »¨ó
`treatment with either concentration of CsA. The only ex-
`½»°¬·±² ©¿ ¬¸¿¬ ¬¸»®» ©¿ ¿ ³»¿² ·²½®»¿» º®±³ ¾¿»ó
`ception was that there was a mean increase from base-
`´·²» ·² ¬¸» ÝÜìó°±·¬·ª» Ì ¸»´°»® ½»´´ °±°«´¿¬·±² º±´´±©ó
`line in the CD4-positive T helper cell population follow-
`·²¹ ðòðëû Ýß ¬®»¿¬³»²¬ò ײ ½±³°¿®·±²ô ¿´´ ½»´´ °±·¬·ª»
`ing 0.05% CsA treatment. In comparison, all cells positive
`º±® ¬¸» ´§³°¸±½§¬·½ ³¿®µ»® ·²½®»¿»¼ º®±³ ¾¿»´·²» º±´ó
`for the lymphocytic markers increased from baseline fol-
`´±©·²¹ ª»¸·½´» ¬®»¿¬³»²¬ò
`lowing vehicle treatment.
`Ú·¹«®» ï ¸±© ¬¸» °»®½»²¬ ½¸¿²¹» º®±³ ¾¿»´·²»
`Figure I shows the percent change from baseline
`º±® ½»´´ »¨°®»·²¹ ¬¸» ´§³°¸±½§¬·½ ³¿®µ»® øÝÜíô ÝÜìô
`for cells expressing the lymphocytic markers (CD3, CD4,
`¿²¼ ÝÜè÷ ¿º¬»® ê ³±²¬¸ ±º ¬®»¿¬³»²¬ º±® ¬¸» ±ª»®¿´´ °¿ó
`and CD8) after 6 months of treatment for the overall pa-
`¬·»²¬ °±°«´¿¬·±²ò Ò±¬» ¬¸¿¬ ¬¸»®» ©¿ ¿ ®»¼«½¬·±² º®±³
`tient population. Note that there was a reduction from
`¾¿»´·²» ·² ¬¸» ²«³¾»® ±º ÝÜíó°±·¬·ª» ½»´´ ·² ¬¸» Ýßó
`baseline in the number of CD3-positive cells in the CsA-
`¬®»¿¬»¼ ¹®±«°ô ©¸·´» ¬¸»®» ©¿ ¿² ·²½®»¿» º®±³ ¾¿»ó
`treated groups, while there was an increase from base-
`´·²» ·² ¬¸» ª»¸·½´»ó¬®»¿¬»¼ ¹®±«°ò ̸»®» ©¿ ¿´± ¿² ·²ó
`line in the vehicle-treated group. There was also an in-
`½®»¿» º®±³ ¾¿»´·²» ·² ¬¸» ²«³¾»® ±º ÝÜìó°±·¬·ª» ½»´´
`crease from baseline in the numbers of CD4-positive cells
`·² ¬¸» ª»¸·½´» ¹®±«°ô ©·¬¸ ¿ ³¿´´»® ·²½®»¿» ·² ¬¸» ðòðëû
`ײ ¹»²»®¿´ô ¬¸»®» ©¿ ¿ ¼»½®»¿» º®±³ ¾¿»´·²» ·² ¬¸» ²«³ó
`in the vehicle group, with a smaller increase in the 0.05%
`In general, there was a decrease from baseline in the num-
`Ýß ¹®±«° ¿²¼ ¿ ´·¹¸¬ ¼»½®»¿» ·² ¬¸» ðòïû Ýß ¹®±«°ò
`¾»® ±º ½»´´ °±·¬·ª» º±® ÝÜíô ÝÜìô ¿²¼ ÝÜè º±´´±©·²¹
`CsA group and a slight decrease in the 0.1% CsA group.
`ber of cells positive for CD3, CD4, and CD8 following
`
`WWW.ARCHOPHTHALMOL.COM
`(REPRINTED) ARCH OPHTHALMOLIVOL 118, NOV 2000
`1491
`ÉÉÉòßÎÝØÑÐØÌØßÔÓÑÔòÝÑÓ
`øÎÛÐÎ×ÒÌÛÜ÷ ßÎÝØ ÑÐØÌØßÔÓÑÔ ñ ÊÑÔ ïïèô ÒÑÊ îððð
`ïìçï
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`J P
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`ATIENT POPULATION
`ÐßÌ×ÛÒÌ ÐÑÐËÔßÌ×ÑÒ
`
`̸» ³»¿² ÍÜ ¿¹» ±º ±«® «¾¶»½¬ ©¿ ëçòð ïíòë §»¿®
`The mean=SD age of our subjects was 59.0 2 13.5 years
`ø®¿²¹»ô îèòèóèìòî §»¿®÷ô ·²½´«¼·²¹ îé ©±³»² ¿²¼ ë ³»²ò
`(range, 28.8-84.2 years), including 27 women and 5 men.
`É·¬¸·² ¬¸· ¹®±«°ô ¬¸»®» ©»®» ïî Ͷ±X ¹®»² ¿²¼ îð ²±²ó
`Within this group, there were 12 Sjogren and 20 non-
`Sj ogren patients.
`Ͷ±X ¹®»² °¿¬·»²¬ò
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`LYMPHOCYTIC MARKERS
`ÔÇÓÐØÑÝÇÌ×Ý ÓßÎÕÛÎÍ
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`©2000 American Medical Association. All rights reserved.
`îððð ß³»®·½¿² Ó»¼·½¿´ ß±½·¿¬·±²ò ß´´ ®·¹¸¬ ®»»®ª»¼ò
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`Figure 5. Immunofluorescence micrographs demonstrating cells positive for the lymphocyte activation marker 0D11a in conjunctival biopsy specimens of
`Ú·¹«®» ëò
`patients with non—Sj6gren keratoconjunctivitis sicca pretreatment and posttreatment with (A and B) 0.05% cyclosporine and (C and 0) vehicle. The number of
`positive cells within epithelium and substantia propria in the cyclosporine-treated group decreased, while the number in the vehicle-treated biopsy sample
`increased (bar=25 pm).
`
`1&4
`ÝÑÓÓÛÒÌ
`
`Ú·¹«®» ë ¿²¼ Ú·¹«®» ê ¸±© ¿ ®»°®»»²¬¿¬·ª» »¬
`Figure 5 and Figure 6 show a representative set
`±º ·³³«²±º´«±®»½»²½» ³·½®±¹®¿°¸ º±® ½»´´ °±·¬·ª» º±®
`of immunofluorescenee micrographs for cells positive for
`¬¸» ³¿®µ»® ÝÜïï¿ ¿²¼ ØÔßóÜÎ º®±³ ¬¸» ²±²óͶ±X ¹®»²
`the markers CD1 1a and HLA-DR from the non-Sjogren
`«¾¹®±«° ¬®»¿¬»¼ ©·¬¸ ðòðëû Ýß ±® ª»¸·½´»ò Ú·¹«®» é
`subgroup treated with 0.05% CsA or vehicle. Figure 1
`¸±© ·³³«²±º´«±®»½»²½» ³·½®±¹®¿°¸ º±® ½»´´ °±·ó
`shows immunofluorescenee micrographs for cells posi-
`¬·ª» º±® ¬¸» ³¿®µ»® ÝÜí ¿²¼ ÝÜïï¿ º®±³ °¿¬·»²¬ ©·¬¸
`tive for the markers CD3 and CD1 1a from patients with
`Ͷ±X ¹®»² ÕÝÍ ¬®»¿¬»¼ ©·¬¸ ðòðëû Ýßò
`Sjogren KCS treated with 0.05% CsA.
`
`̸»» º·²¼·²¹ °®±ª·¼» ¿¼¼·¬·±²¿´ »ª·¼»²½» ¬¸¿¬ ·²ó
`These findings provide additional evidence that in-
`º´¿³³¿¬·±² °´¿§ ¿ ®±´» ·² ¬¸» °¿¬¸±¹»²»· ±º ÕÝÍ ¿²¼
`flammation plays a role in the pathogenesis of KCS and
`«¹¹»¬ ¬¸¿¬ ³±¼«´¿¬·²¹ ¬¸» «²¼»®´§·²¹ ·³³«²» ®»ó
`suggests that modulating the underlying immune re-
`°±²» ³¿§ °®±ª» ³±®» »ºº·½¿½·±« ·² ¬¸» ¬®»¿¬³»²¬ ±º
`sponse may prove more efficacious in the treatment of
`ÕÝÍ ¬¸¿² ¬¸» º®»¯«»²¬ «» ±º ¿®¬·º·½·¿´ ¬»¿®ò ̱°·½¿´ Ýß
`KCS than the frequent use of artificial tears. Topical CsA
`¸¿ ¾»»² «½½»º«´´§ «»¼ º±® ¬¸» ¬®»¿¬³»²¬ ±º ½¿²·²»
`has been successfully used for the treatment of canine
`¼®§ »§» º±® ³¿²§ §»¿®ò ͬ«¼·» ·² ¬¸» ½¿²·²» ÕÝÍ ³±¼»´
`dry eye for many years. Studies in the canine KCS model
`¸¿ª» ¼»³±²¬®¿¬»¼ ¬¸¿¬ Ýß ¼»½®»¿» ¬¸» ½±²¶«²½¬·ª¿´
`have demonstrated that CsA decreases the conjunctival
`¿²¼ ´¿½®·³¿´ ¹´¿²¼ ´§³°¸±½§¬·½ ·²º·´¬®¿¬»òîìóîê
`and lacrimal gland lymphocytic infiltrates.2“'25
`ر©»ª»®ô ¬¸»®» ¸¿ª» ¾»»² ±²´§ ¿ ´·³·¬»¼ ²«³¾»® ±º
`However, there have been only a limited number of
`®»°±®¬ ±² ¬¸» «» ±º ¬±°·½¿´ Ýß ·² ¬¸» ¬®»¿¬³»²¬ ±º ¼®§
`ײ ¬¸» °®»»²¬ ¬«¼§ô ·³³«²±¸·¬±½¸»³·½¿´ ¿²¿´§· ©¿
`reports on the use of topical CsA in the treatment of dry
`In the present study, immunohistochemical analysis was
`»§» §²¼®±³» ·² ¸«³¿²îéóîç ©·¬¸ ±²´§ ï ¿¬¬»³°¬ ¬± ´±±µ
`«»¼ ¬± »ª¿´«¿¬» ½¸¿²¹» ·² ¬¸» °®»»²½» ±º ½»´´ °±·ó
`used to evaluate changes in the presence of cells posi-
`eye syndrome in humans27'29 with only 1 attempt to look
`at the effect of the treatment at a cellular level.” Power
`¿¬ ¬¸» »ºº»½¬ ±º ¬¸» ¬®»¿¬³»²¬ ¿¬ ¿ ½»´´«´¿® ´»ª»´òíð б©»®
`¬·ª» º±® ´§³°¸±½§¬·½ ¿²¼ ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µó
`tive for lymphocytic and lymphocyte activation mark-
`»¬ ¿´íð ®»°±®¬»¼ ¿ ·¹²·º·½¿²¬ ®»¼«½¬·±² ·² ÝÜìó°±·¬·ª»
`»® ·² ½±²¶«²½¬·ª¿´ ¾·±°§ °»½·³»² ±º °¿¬·»²¬ ©·¬¸ ³±¼ó
`ers in conjunctival biopsy specimens of patients with mod-
`et al3° reported a significant reduction in CD4-positive
`Ì ´§³°¸±½§¬» ·² ¾±¬¸ ¬¸» ½±²¶«²½¬·ª¿´ »°·¬¸»´·«³ ¿²¼
`»®¿¬» ¬± »ª»®» ÕÝÍô º±´´±©·²¹ ¬®»¿¬³»²¬ ©·¬¸ ðòðëû Ýßô
`T lymphocytes in both the conjunctival epithelium and
`erate to severe KCS, following treatment with 0.05% CsA,
`0.1% CsA, or vehicle. We found that CsA treatment re-
`¬¸» «¾¬¿²¬·¿ °®±°®·¿ ±º °¿¬·»²¬ ©·¬¸ »½±²¼¿®§ Ͷ±X ó
`ðòïû Ýßô ±® ª»¸·½´»ò É» º±«²¼ ¬¸¿¬ Ýß ¬®»¿¬³»²¬ ®»ó
`the substantia propria of patients with secondary Sjo-
`¹®»² §²¼®±³» ½±³°¿®»¼ ©·¬¸ ²±²o¼®§ »§» ½±²¬®±´ º±´ó
`¼«½»¼ ¬¸» ²«³¾»® ±º ¿½¬·ª¿¬»¼ Ì ´§³°¸±½§¬» ©·¬¸·² ¬¸»
`gren syndrome compared with non—dry eye controls fol-
`duced the number of activated T lymphocytes within the
`´±©·²¹ ¬®»¿¬³»²¬ ©·¬¸ Ýßò ̸» °®»»²¬ ¬«¼§ ¿´± ¼»³ó
`±½«´¿® «®º¿½» ±º °¿¬·»²¬ ©·¬¸ ¿²¼ ©·¬¸±«¬ Ͷ±X ¹®»² §²ó
`lowing treatment with CsA. The present study also dem-
`ocular surface of patients with and without Sj ogren syn-
`drome. After 6 months of treatment with 0.05% CsA, sta-
`±²¬®¿¬»¼ ¿ ·¹²·º·½¿²¬ ¼»½®»¿» ·² ÝÜíó°±·¬·ª» ½»´´ ¿º¬»®
`¼®±³»ò ߺ¬»® ê ³±²¬¸ ±º ¬®»¿¬³»²¬ ©·¬¸ ðòðëû Ýßô ¬¿ó
`onstrated a significant decrease in CD3-positive cells after
`ê ³±²¬¸ ±º ðòðëû Ýß ¬®»¿¬³»²¬ ·² °¿¬·»²¬ ©·¬¸ Ͷ±X ó
`¬·¬·½¿´´§ ·¹²·º·½¿²¬ ¼»½®»¿» ©»®» »»² ·² ½»´´ °±·¬·ª»
`6 months of 0.05% CsA treatment in patients with Sjo-
`tistically significant decreases were seen in cells positive
`¹®»² §²¼®±³»ò
`º±® ÝÜïï¿ ¿²¼ ØÔßóÜÎ ½±³°¿®»¼ ©·¬¸ ¬¸±» ·² ª»¸·½´»
`gren syndrome.
`for CD1 1a and HLA-DR compared with those in vehicle
`Ú«®¬¸»®³±®»ô ¬¸» ²«³¾»® ±º ½»´´ °±·¬·ª» º±® ÝÜïï¿
`º±® ¬¸» ±ª»®¿´´ °¿¬·»²¬ °±°«´¿¬·±²ò É·¬¸·² ¬¸» Ͷ±X ¹®»² °¿ó
`Furthermore, the number of cells positive for CD1 1a
`for the overall patient population. Within the Sjogren pa-
`¿²¼ ØÔßóÜÎô ©¸·½¸ ¿®» ´§³°¸±½§¬» ¿½¬·ª¿¬·±² ³¿®µó
`¬·»²¬ «¾¹®±«° ¬®»¿¬»¼ ©·¬¸ ðòðëû Ýßô ¬¸»®» ©»®» ¿´±
`and HLA-DR, which are lymphocyte activation mark-
`tient subgroup treated with 0.05% CsA, there were also
`»®ô ¼»½®»¿»¼ ·¹²·º·½¿²¬´§ ·² °¿¬·»²¬ °±°«´¿¬·±² ¬®»¿¬»¼
`·¹²·º·½¿²¬´§ ¹®»¿¬»® ¼»½®»¿» ¬¸¿² ©·¬¸ ª»¸·½´» ·² ¬¸»
`ers, decreased significantly in patient populations treated
`significantly greater decreases than with vehicle in the
`©·¬¸ Ýßò ØÔßóÜÎ · ¿ ½´¿ ×× ³¿¶±® ¸·¬±½±³°¿¬·¾·´·¬§
`²«³¾»® ±º ½»´´ °±·¬·ª» º±® ÝÜí ¿²¼ ÝÜïï¿ò
`with CsA. HLA-DR is a class 11 major histocompatibility
`number of cells positive for CD3 and CD11a.
`
`WWW.ARCHOPHTHALMOL.COM
`(REPRINTED) ARCH OPHTHALMOLIVOL 118, NOV 2000
`14-93
`ÉÉÉòßÎÝØÑÐØÌØßÔÓÑÔòÝÑÓ
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`©2000 American Medical Association. All rights reserved.
`îððð ß³»®·½¿² Ó»¼·½¿´ ß±½·¿¬·±²ò ß´´ ®·¹¸¬ ®»»®ª»¼ò
`Downloaded From: http://archopht.jamanetwork.com/ by a University of Michigan User on 01/25/2016
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`Figure 6. lmmunofluorescence micrographs demonstrating cells positive for HLA-DR in con/unct/val biopsy specimens of patients with non—S/tigren
`Ú·¹«®» êò
`keratoconjunctivitis sicca pretreatment and posttreatment with (A and B) 0.05% cyclosporine and (C and D) vehicle. A decrease in the number of positive cells
`Within epithelium and substantia propria in the 0.05% cyclosporine-treated group is apparent compared with an increase in number in the vehicle-treated biopsy
`sample. E and F, Example of a negative control fora vehicle biopsy in which the primary antibody was omitted. Bar=25 pm (A-0).
`
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`complex antigen that is expressed in inflamed regions and
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`serves as a ligand for the T—cell receptor. CD4+ T lym-
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`phocytes are activated through a signal from HLA—DR mol-
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`ecules of antigen—presenting cells.” Immunopathologic
`studies show evidence of immune activation of the con-
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`junctival epithelium in Sj ogren syndrome. Compared with
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`control eyes, a significantly greater percentage of con-
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`junctival epithelial cells from patients with Sjog